(287h) Controlled Release of Inflammasome Modulators to Promote Tissue Repair

Purpose/Objectives: The inflammasome is a multiprotein complex that is involved in the core innate immune response to sterile and infected injury. Appropriate activation of the inflammasome is essential for initiation of healing in the skin and in other tissues. In comorbidities associated with poor wound healing, such as diabetes, the inflammasome undergoes a robust and sustained activation with delayed or failed resolution that prevents the wound from progressing through the inflammatory phase into the proliferative phase of healing. As a readily druggable pathway, the inflammasome is thus an ideal target for modulating diabetic wound repair by release of small molecule inhibitors from wound dressings, thereby limiting the negative effects of sustained and dysregulated inflammasome activation and allowing a wound to progress towards resolution. Extracellular ATP released from damaged or necrotic cells is considered a damage-associated molecular pattern (DAMP). Detection of extracellular ATP by P2X₇ purinergic receptors result in the opening of membrane channels and the rapid flux of potassium and calcium ions across cellular membranes. The dramatic loss of potassium can result in the activation of the NLRP3 inflammasome, primarily in macrophages in wounds but in other cells as well. We have previously shown that the competitive P2X₇ receptor antagonist A438079 is a robust inhibitor of inflammasome activation by preventing potassium and calcium ion flux, and mitochondrial ROS generation upstream of NLRP3 inflammasome assembly, IL-1b processing and release, and pyroptotic cell death. Here, we investigate the generation of a controlled release scaffold for loading and release of A438079 from insoluble silk fibroin scaffolds for use as an inflammasome-modulating wound dressing.

Methodology: Silk fibroin was isolated from Bombyx mori silkworm cocoons by standard methods. Films of differing thicknesses were prepared by casting of different volumes of 8% silk fibroin solution into 100-mm petri dishes followed by solvent evaporation at room temperature. Circular films with a diameter of 6 mm were prepared with a hollow punch. Films were made insoluble by autoclaving. Film thickness was measured by digital micrometer and rheological properties were measured by dynamic mechanical analysis (DMA). Film degradation was determined by incubation in PBS for 40 days and measuring material loss as a factor of film thickness via digital micrometer and protein release by BCA assay. UV-VIS spectroscopy analysis of A438079 was performed and diagnostic wavelengths demonstrating linear absorbance versus concentration were identified at 226 nm and 258 nm. A438079, a water-insoluble drug solubilized with 50% DMSO, was loaded into empty films overnight by passive uptake. Loaded films were dried and stored before further use. Film release into PBS was measured for 5 days and quantified by UV-VIS spectroscopy. Released drug was tested for inflammasome inhibition by treating control or LPS-primed J774-DUAL mouse macrophages in media pre-incubated with loaded films and inducing IL-1beta release by exposure to extracellular ATP. IL-1beta release was measured by ELISA assay. Excisional wound healing studies were performed in db/db mice. IL1b levels were measured by immunohistochemistry.

Results: Silks films of tunable thickness were prepared and made insoluble, with no evidence of swelling or degradation over a period of 40 days. Drug release followed an exponential plateau (R²=0.82 to 0.98) and achieved a maximum cumulative release of approximately 200 µM A438079 from the 55 µm-thick films or approximately 360 µM A438079 from 100 µm-thick films at 3 days. Release drug was tested against J774-DUAL macrophages primed with LPS and stimulated with extracellular ATP. Conditioned media from 4 hours of drug release exhibited nearly completely inhibition of IL-1b release and phenotypic evidence of pyroptosis, indicating robust inhibition of inflammasome activation in vitro. In vivo evidence supported a dramatic inhibition of inflammasome levels in rapidly healing diabetic wounds treated with silk films loaded with A438079.

Conclusion/Significance: These studies demonstrate the controlled, tunable release of small molecule inflammasome modulators from a biocompatible drug delivery scaffold. In vivo studies evaluated the potential for accelerating wound healing in a full-thickness splinted model. The inflammasome is a druggable, but under-studied factor in impaired, diabetic wound healing. A novel approach to inhibiting the dysregulated inflammasome activity in wounds by controlled release of upstream receptor modulators, thereby preventing the release of damaging, sustained inflammatory mediators may help to control healing and promote repair.